The traditional way for recovering an intracellular POI, such as an enzyme, has been to use a mechanical disruption method (Naglak et al 1990) such as a bead mill or a cell homogenizer operating with a french press principle. However, these mechanical disruption methods suffer from the disadvantage that they are energy consuming methods with a low capacity and the cell homogenizers or similar equipment required for mechanical disruption are expensive to purchase. In addition, mechanical methods expose the cells, and hence the extracted POI to very harsh conditions, especially as most proteins will be denatured by the heat generated unless the mechanical device and/or homogenate is efficiently cooled.
Furthermore, some cells, such as yeast cells (such as those from Hansenula) are difficult to disrupt mechanically and more than one passage through a cell homogenizer is needed. The cell homogenate may also contain cell wall fragments and DNA, which results in a high viscosity. This means that the separation of cell debris from the POI can prove to be a difficult operation. In addition, the resulting cell free homogenate may contain not only the intracellular POI but also a large number (sometimes several thousand) of different intracellular proteins and enzymes associated with the general cell metabolism. This means that the resultant cell free homogenate may be not only difficult to concentrate by ultrafiltration but may also provide problems with respect to obtaining the right commercial concentration of a given POI.
In order to minimise the potential detrimental effect of some mechanical disruption methods, chemical methods using, for example, detergents have been developed to permeabilize yeast cells. By way of example, the non-ionic detergent, polyethoxylated octylphenols, commercially available as Triton X-100, has been used either alone or in combination with freeze thaw cycles (referenced in Naglak et al 1990). In addition, U.S. Pat. No. 5,124,256 (Crahay et al 1992) discloses a method whereby proteins were extracted from Saccharomyces yeast by means of treating the yeasts in an aqueous medium with a neutral water-soluble mineral salt and a non-ionic water-soluble polyethoxylated alkylphenol surfactant having a Hydrophilic Lipophilic Balance (HLB) of between 8 and 15.
However, these non-ionic water-soluble polyethoxylated alkylphenol surfactants which include polyethoxylated octylphenols, nonylphenols and tributylphenols, (particularly those commercially available under the trade marks TritonX-100, Nonidet P-40 and Sapogenat T-080) suffer from the drawbacks that (i) they may not have a significant extracting effect when used alone and (ii) these surfactants can interfere with subsequent measurements of the enzymatic activity of the POI.
Several organic solvents have also been used to both permeabilize yeast cells in in situ enzymatic assays and for removing proteins from yeast cells. Examples of such solvents include but are not limited to toluene, ethyl acetate, dimethyl sulfoxide, and benzene combined with glycerol (Naglak et al 1990). However, these solvents are unattractive to use in industrial scale production when fermentor volumes of up to 200 m3 are required.
Digitonin and other naturally occuring saponins have also been shown to permeabilize a number of eukaryotic cells (see Joshi et al 1989). Although the exact mechanism of digitonin permeabilization is not known, it is believed that digitonin forms a complex with the cholesterol present in the cell membrane and renders the membrane leaky. Digitonin permeabilization of yeast cells may also be due to the complexing of ergosterols of the yeast membrane. Joshi et al (1989) used digitonin (0.1%) to permeabilize the yeast Kluyveromyces which facilitated the intracellular catalysis of lactose to glucose and galactose. The non-ionic detergent saponin, from Quillaja Bark, is another cholesterol complexing agent, which is known to permeabilise at least mammalian cells (Naglak et al 1990). Again, like the non-ionic detergents as outlined above, the use of digitonin and other naturally occuring saponins may suffer from the drawback that when used alone, they may not have a significant extracting effect.
U.S. Pat. No. 5,240,834 (Frankel et al) describes a protein extraction using the detergent Sarkosyl (N-lauryl sarcosine), see Example 1 (paragraphs 3 to 4) as well as lines 67 of column 3 to line 2 of column 4. U.S. Pat. No. 6,251,626 (Stougaard et al) describes extraction of HOX from yeast or bacterial cells, but the protein is released by mechanical disruption in a French press. The yeast cells are exposed to enormous pressure (to 20,000 p.s.i.) in order to disrupt them and to release the recombinant HOX enzyme (lines 25 to 31 of column 40).
Chaotropic agents have also been used to faciliate the extraction of intracellular enzymes. By way of example, U.S. Pat. No. 3,801,461 (Miyake and Shiosaka 1974) discloses a process for extracting intracellular enzymes produced in the mycelia or cells of fungi or bacteria using a chaotrophic solution such as a urea solution. U.S. Pat. No. 4,683,293 (Craig 1987) also discloses a method for selective extraction of lipophilic proteins from transformed cells of the genus Pichia by cell lysis in the presence of chaotrophic salts such as sodium thiocyanate, sodium iodide, sodium hypochlorite, lithium bromide, guanidium hydrochloride and urea. However, chaotrophic agents suffer from the disadvantage that exposure of the POI to a chaotrophic agent, such as urea can result in an actual loss of enzyme activity through denaturation of the POI.
In addition to the drawbacks cited above, the above cited prior art only relates to the permeabilisation of host cells to low molecular weight molecules while the POI remains unchanged within the cell. In particular, none of the above cited prior art relates to the extraction of a membrane associated intracellular POI under conditions which do not affect the nature and/or activity of the POI. More in particular, none of the above cited prior art relates to a method for assisting in the release of a membrane associated intracellular POI which is trapped and is incapable of being secreted from a host cell.
The present invention thus seeks to overcome the problems associated with the extraction methods of the prior art.
The present invention thus provides a method for releasing a soluble or membrane associated intracellular protein of interest (POI) from a host organism.